Inhibition of Amyloid-β Fibril Formation

Monomerization of Aβ₄₂ (Adelab Scientific) was prepared by NaOH treatment as described previously [65 (link)] whereas mature Aβ₄₂ fibrils were prepared at 37°C for 24 h with shaking (50 rpm). The ability of various inhibitors to prevent Aβ₄₂ fibril formation was assessed using an in situ thioflavin-T (ThT) (20 µM) assay. Non-monomerized Aβ₄₂ (100 µM) was examined across a range of molar ratios (1 : 1, 1 : 2, 1 : 10 and 1 : 20) (Aβ₄₂:inhibitor) in the absence and presence of inhibitors. Assays were performed in PBS (pH 7.4) and 1% DMSO at 35°C in quiescence. The ability of the inhibitors to prevent Aβ₄₂ fibrilization was determined by comparing the ThT fluorescence at the conclusion of each assay. [66 (link)] The inhibition of primary-nucleation mediated Aβ₄₂ aggregation was also monitored using an in situ ThT assay where monomerized Aβ₄₂ (10 µM) was incubated in the absence and presence of inhibitors under conditions stated above at 25°C. All assays were performed using a FLUOstar Optima plate reader (BMG Lab Technologies) with excitation and emission wavelengths set at 440 nm and 490 nm, respectively. All assays were performed at least three times and data are reported as mean ± SEM of three independent assays.